Resveratrol (trans-3,4′,5-trihydroxystilbene) and/or its corresponding glucoside (piceid) are stilbene compounds reported to have many beneficial health effects. Resveratrol is a potent antioxidant, decreasing low density lipid (LDL) oxidation, a factor associated with the development of atherosclerosis (Manna et al., J. Immunol., 164:6509-6519 (2000)). It is also reported to lower serum cholesterol levels and the incidents of heart disease. This effect as been attributed to a phenomenon known and the “French Paradox”. French citizens that regularly consume red wine tend to have lower incidents of heart disease and serum cholesterol levels even though this same group tends to consume foods high in both fat and cholesterol. There is also evidence that resveratrol may have other cardiovascular protective effects including modulation of vascular cell function, suppression of platelet aggregation, and reduction of myocardial damage during ischemia-reperfusion (Bradamante et al., Cardiovasc. Drug. Rev., 22(3):169-188 (2004)). Resveratrol is reported to have anti-inflammatory effects associated with the inhibition of the cyclooxygenase-1 (Cox-1), an enzyme associated with the conversion of arachidonic acid to pro-inflammatory mediators. It may also aid in the inhibition of carcinogenesis (Schultz, J., J Natl Cancer Inst., 96(20):1497-1498 (2004); Scifo et al., Oncol Res., 14(9):415-426 (2004); and Kundu, J. and Surh, Y., Mutat Res., 555(1-2):65-80 (2004)).
Resveratrol is classified as a phytoalexin due to its antifungal properties. It appears that some plants produce resveratrol as natural defense mechanism against fungal infections. For example, red grapes have been reported to produce resveratrol in response to fungal infections. Fungal cell wall components can stimulate local expression of the resveratrol synthase gene in grapes. The antifungal property of resveratrol has been applied to plants that do not naturally produce the compound. Transgenic plants modified to express the resveratrol synthase gene exhibit improved resistance to fungal infections. Furthermore, it has been reported that treatment of fresh fruits and vegetables with an effective amount of resveratrol will significantly increase shelf life (Gonzalez-Urena et al., J. Agric. Food Chem., 51:82-89 (2003)).
Use of resveratrol in commercial products (e.g., pharmaceuticals, personal care products, antifungal compositions, antioxidant compositions, dietary supplements, etc.) is limited due to the current market price of the compound. Methods to extract resveratrol from plant tissues such as red grape skins, peanuts or the root tissue of Polygonum cuspidatum are not economical. Means to produce resveratrol by chemical synthesis are difficult, inefficient, and expensive. There is a need for an efficient and cost effective method to synthesize resveratrol.
Resveratrol and/or resveratrol glucoside are naturally produced in a variety of herbaceous plants (Vitaceae, Myrtaceae, and Leguminosae). The resveratrol biosynthesis pathway is well known. In plants, a single type III polyketide synthase (resveratrol synthase; E.C. 2.3.1.95) catalyzes three consecutive Claisen condensations of the acetate unit from malonyl CoA with the phenylpropanoid compound p-coumaroyl CoA, which is succeeded by (1) an aldol reaction that forms the second aromatic ring, (2) cleavage of the thioester, and (3) decarboxylation to form resveratrol.
Industrial microbial production offers a possible means to economically produce commercial quantities of resveratrol. Microbial production requires functional expression of the resveratrol synthase gene in the presence of suitable quantities of malonyl CoA and p-coumaroyl CoA. Cost-effective microbial production generally requires host cells having the ability to produce both malonyl CoA and p-coumaroyl CoA in suitable quantities from a relatively inexpensive carbon substrate.
Many naturally occurring microorganisms, such as E. coli and Saccharomyces cerevisiae, produce malonyl CoA, albeit in relatively low quantities ranging from barely detectable levels up to about 30 μM (Davis et al., J. Biol. Chem., 275:28593-28598 (2000) and Subrahmanyam, S, and Cronan, J., J. Bacteriol., 180:45964602 (1998)). Since malonyl CoA is involved in fatty acid biosynthesis, a host cell capable of synthesizing significant amounts of oil (e.g., an oleaginous microorganism) may produce suitable quantities of malonyl CoA (or may exhibit the ability to accommodate high-flux malonyl CoA production).
Recombinant microbial production of resveratrol also requires the substrate p-coumaroyl CoA. This phenylpropanoid compound is ubiquitously produced in plants, but is found in relatively low quantities (if at all) in many microbial host cells. As such, the microbial host cell selected for resveratrol production should be engineered to produce p-coumaroyl CoA.
The enzyme coumaroyl CoA ligase (E.C. 6.2.1.12) catalyzes the conversion of para-hydroxycinnamic acid (pHCA) to p-coumaroyl CoA. In the past, coumaroyl CoA ligases were generally considered to only exist in plants, however a coumaroyl CoA ligase was recently reported in the filamentous bacterium Streptomyces coelicolor (Kaneko et al., J. Bacteriol., 185(1):20-27 (2003)). Recombinant microbial expression of coumaroyl CoA ligase has been reported (Becker et al., FEMS Yeast Research, 4(1):79-85 (2003)); Keneko et al., supra; Watts et al., Chembiochem, 5:500-507 (2004); and Hwang et al., Appl. Environ. Microbiol., 69(5):2699-2706 (2003)).
Recombinant biosynthesis of coumaroyl CoA requires a suitable source of pHCA. The source of pHCA may be supplied exogenously to the host cell or it may be produced within the host cell. Preferably, the host cell can be engineered to produce suitable levels of pHCA when grown on an inexpensive carbon source, such as glucose. Recombinant microbial host cells engineered to produce and/or accumulate phenylpropanoid-derived compounds (I.e., p-hydroxycinnamic acid) have been reported (U.S. Pat. Nos. 6,368,837, 6,521,748, U.S. application Ser. Nos. 10/138,970, 10/439,479, 10/621,826; and Schroder, J. and Schroder, G., Z. Naturforsch, 45:1-8 (1990)). Recombinant expression of a coumaroyl CoA ligase in cells engineered to produce para-hydroxycinnamic acid (pHCA) results in the production of p-coumaroyl CoA (p-coumaric acid).
Microbial expression of enzymes involved in the phenylpropanoid pathway to produce the flavanone narigenin is described by Watts et al. (supra) and Hwang et al. (supra). Specifically, Watts et al. describe the simultaneous expression of a phenylalanine ammonia lyase, a tyrosine ammonia lyase, a cinnamate 4-hydroxylase (C4H), a coumaroyl CoA ligase, and a chalcone synthase (E.C. 2.3.1.74) in E. coli to produce narigenin and phloretin up to 20.8 mg/L. However, Watts et al. were not able to actively express cinnamate-4-hydroxylase (C4H) in E. coli and had to supply exogenous p-coumaric acid or 3-(4-hydroxyphenyl)propionic acid to obtain significant concentrations of the desired products. Watts et al. do not describe recombinant microbial production of resveratrol.
Hwang et al. describe recombinant bacterial (E. coli) production of the flavanones pinocembrin and narigenin by simultaneously expressing phenylalanine ammonia lyase, coumaroyl CoA ligase, and a chalcone synthase (E.C. 2.3.1.74). The bacterial coumaroyl CoA ligase used by Hwang et al. was able to convert both cinnamic acid to cinnamoyl CoA and p-coumaric acid to p-coumaroyl CoA, resulting in the production of pinocembrin (from phenylalanine) and naringenin (from tyrosine) as the PAL used also exhibited tyrosine ammonia lyase activity, resulting in the production of pHCA. In the absence of exogenously supplementing the medium with excess L-phenylalanine and/or L-tyrosine, only small amounts of each flavanone are produced (<0.3 μg/L). Hwang et al. do not describe recombinant microbial production of resveratrol.
Becker et al. (supra) describe recombinant expression of several phenylpropanoid pathway genes in Saccharomyces cerevisiae FY23 for the production of resveratrol. Genes encoding a coumaroyl CoA ligase and a resveratrol synthase were recombinantly expressed in S. cerevisiae in a culture medium supplemented with pHCA, producing resveratrol in amounts up to 1.45 μg/L in the culture volume. Becker et al. report that experiments supplementing the culture medium with additional precursors necessary for resveratrol production do not produce significantly more resveratrol. Becker et al. do not illustrate a method to produce significant quantities of resveratrol in a recombinant host cell, including production of resveratrol from a commonly used (and economical) fermentable carbon source (e.g., glucose).
The problem to be solved is to provide a method for recombinant microbial production of resveratrol in significant amounts.